This renewal of the P01 is a logical extension of progress made during the current cycle and leverages the substantial contributions in this field made by the PIs. During this cycle, we identified an AAV capsid clinical candidate - AAV8 - to use for liver applications. This was evaluated in mouse models of homozygous familial hypercholesterolemia (hoFH) and was shown to be highly effective and safe. We accelerated our clinical development plan of an AAV-LDLR product and reached out to the Food and Drug Administration and met with the Recombinant DNA Advisory Committee, both of whom were supportive of moving forward with a Phase 1 trial. In preparation for this, we completed IND-enabling studies and are ready to file an IND. The centerpiece of the competing renewal is Project 1 that contains a phase 1 clinical trial of AAV8-LDLR in subjects with hoFH. Projects 2 and 3 build on our pre-clinical experiences with AAV8 in animal models of hoFH in several ways. We plan to conduct proof-of-concept studies in mouse models to develop AAV8 gene therapy for lecithin- cholesterol acyltransferase (LCAT) deficiency which is an inherited dyslipidemia associated with low HDL and renal disease. This will be followed by IND-enabling studies with AAV8-LCAT with the goal of having an approved IND for LCAT deficiency by the completion of the competing renewal. Gene therapy for hoFH and LCAT deficiency has some important similarities that will greatly facilitate the translation of LCAT gene therapy in the clinic although there are differences that provide scientific rationale for pursuing both. The target organ pathology differs in that hoFH causes atherosclerosis while LCAT deficiency leads to renal disease. Furthermore, LDLR confers a cell autonomous function in which the percent hepatocyte transduction is more important than the overall level of LDLR expression. This contrasts with LCAT which is a secreted protein with effects beyond the cells that are transduced; efficacy is more related to overall expression of the enzyme rather than the number of transduced cells. We also will conduct studies to improve gene therapy for hoFH in ways that are specific to this disease - introducing gain-of-function mutations into LDLR to increase vector potency - and using approaches that will be of potential value for all applications of liver directed gene therapy - creating variants of AAV3B that escape antibody neutralization and allow for efficient in vivo gene transfer in the presence of AAV8 neutralizing antibodies present from natural infections or from an earlier treatment with an AAV8 vector. Two of the three projects in the current grant will be retained (PIs: Dan Rader and James Wilson) as will the three scientific cores (Vector, Animal Models and Cell Morphology). The project currently directed by Guangping Gao (UMass) on genotoxicity of AAV genomes will be replaced by a project directed by Marina Cuchel (Penn) to conduct the hoFH clinical trial and LCAT IND-enabling studies. (End of Abstract) INDIVIDUAL PROJECTS AND CORE UNITS PROJECT 1:CLINICAL DEVELOPMENT OF GENE THERAPY FOR FH AND LCAT DEFICIENCY (Cuchel, Marina)